Production of riboflavin by ashbya gossypii



United States Patent Ofiice 2,702,265 Patented Feb. 15, 1058 PRODUCTION OF RIDOFLAVIN BY ASHIYA GOSSYPII Karl L. Smiley and Leonard Stone, 1' Ill, Illinois tofllramwalkerdrsonslnc l'eorimlll.

N D Appllefliuuoctober 1953,

o serlal N- SM 2,

45 Claim. (Cl. 195-38) This application is a continuation-impart of our prior application Serial No. 259,714, filed December 3, 1951, which is a continuation-in-part of our application Serial No. 770,330, filed August 23, 1947. v

The present invention relates to the biological synthesis of riboflavin in connection with the cultivation or propagation of the ascomycete Ashbya gossypii. More particularly, it relates to means for increasing or augmenting the yields of riboflavin produced by this organism not only in media'in which it normally produces substantial yields of riboflavin, but also in media in which it otherwise propagates with yields of riboflavin which are negligible or practically insignificant, i. e., so small that its recovery is not practicable. The invention contemplates not only procedures aimed at the production of riboflavin-containing mashes or media from which riboflavin may be recovered in a greater or less degree of purity, as desired, but also at production of such mashes or media which may be dried, with or without prior separation of extraneous material, to secure products containing, in addition to riboflavin, associated vitamins and animal growth factors, for use as animal feed supplements.

Ashbya gossypii, the organism which is employed in our process, is related to, but distinct from, Eremothecium ashbyil, the riboflavin-producing ability of which is well known. Guilliermond and co-workers have pointed out both morphological and physiological characteristics which serve to differentiate the two organisms. According to these investigators Ashbya gossypii exists almost exclusively in the mycelial state, whereas in Eremothecium ashbyii the budding form predominates. Likewise, with the former, cells which give rise to ascospores are plurinucleate rather than mononucleate as in Eremothecium ashbyii. The difference in shape between vegetative spores of the two organisms also serves to distinguish them. Of greater significance in the present instance is the observation of Schopfer, subsequently confirmed by other investigators, that Ashbya gossypii, in media in which Eremorhecium ashbyil produces substantial yields of riboflavin, is incapable of producing more than traces of riboflavin. Thus, whereas with E'remothecium ashbyii, substrates of widely varying composition may be utilized successfully for production of riboflavin, it has been our experience as well as that of others that the requirements of substrates for securing appreciable yields of riboflavin in the propagation of Ashbya gossypii are quite limited and specific. I

In accordance with the present invention we have found that in media capable of supporting the growth of the organism Ashbya gossypii, including media in which the yields of riboflavin have been negligible or practically insignificant, substantial yields of riboflavin may be secured and the synthesis of riboflavin greatly augmented by supplementing the medium with a propionate radicalyielding compound of the class consisting of propiomc acid, its calcium, alkali metal and ammonium salts, its lower alkyl esters and its aryl and aryl-alkyl esters with not more than 9 carbon atoms. When such compounds are present in the medium in molar concentrations of from about 0.001 to 0.03, riboflavin production proceeds more rapidly and final yields in a given period are substantially increased and in many cases greatly multiplied over those in any comparable media lacking the propionateyielding compound. Furthermore, we have also found that when brewer's yeast or yeast substance-containing material, such as yeast extract or yeast cell substance, is added with or is present in conjunction with a propionate radicalyielding compound as referred to above, a markedly greater Increase in yield of riboflavin is secured over that produced m the same medium without the presence of the yeast or yeast substance.

As will more fully hereinafter appear, our process may be employed to fortify the constituents of the medium or mash tn which the organism is propagated, for example, fermentation residues or other proteinaccous grain processtng residues, with riboflavin, or if desired, more potent concentrates, or even the crystalline vitamin, can be prepared. Over and above riboflavin, this organism elaborates certain as yet unidentified factors which are important in ultry and animal nutrition. Therefore, the method which has been developed in accordance with our nvention is especially suitable for manufacturing adluncts to animal feeds.

The fermentative process may be conducted generally in the manner of that described in United States Patent bio. 2,595,827 of C. S. Borufi et al., granted May 6, 1952, In which the nutrient content of stillage from grain alcohol fermentatiqns is enhanced by refermentation. In this procedure, gram alcohol thin stillage is adjusted to the proper reaction for subsequent fermentation by the additron of alkali. The medium is then preferably supplied with carbohydrate and/ or assimilable nitrogen and is sterilized. After cooling, the medium is inoculated with Ashbya gossypii an d agitated mechanically and aerated whlle bemg maintained at the optimum temperature for vitamin production. After refermentation the liquor can be concentrated and dried in any one of several ways well known in the art.

In carrying out the present invention, the basal medium or substrate employed is one in which the organism Ashby: gossypii may be effectively propagated, even with neglrglbleor practically insignificant yields of riboflavin, although it IS preferred to employ media or substrates in which substantial yields of riboflavin are secured. We incorporate m the medium a propionate radical-yielding compound, as hereinafter more fully set forth, and which we have found to guide the activity of the organism in the direction of riboflavin biosynthesis. The presence of such compounds causes the organism to produce substantial yields of riboflavin even in media in which, in the absence of the propionate radical-yielding compound, the organism produces an inappreciable or practically negl' ble yield of riboflavin; and in media in which substantial yields of riboflavin are otherwise produced, the presence of such compounds greatly augments the yield of riboflavin synthesized by the organism. As will be more fully set forth hereinafter, the presence of both yeast substancecontaining materials and a propionate radical-yielding aompound even more greatly enhances the yield of riboavin.

In preparing the medium in the practice of the invention, we incorporate in the medium a proteinaccous material of plant or animal origin, which may be hydrolyzed, partly hydrolyzed or unhydrolyzed, although generally the presence of at least a minor proportion of protein hydrolysis products is preferred, with say at least 5% of the protein nitrogen represented by protein hydrolysis roducts. The hydrolysis may be efiected by the action 0 acid or enzymes by the customary procedures known in the art. Thus we may employ plant proteinaccous materials, such as zein, wheat gluten, hordein, soybean casein, or the like; grain meals and protein containing products derived from grains, such as corn meal, ground wheat, oats, milo,

arley or the like, soybean meal, linseed meaI, corn germ.

meal, wheat germ, wheat middlings, red dog flour or the like, and plant proteinaccous materials which have been at least partly hydrolyzed by acid or enzyme action, such as soybean peptone, grain mashes which have been subjected to the proteolytic action of enzymes such as those of barley malt, and proteinaccous grain processing residue, such as corn steep liquor, grain alcohol fermentation stillage, acetonc-butyl alcohol fermentation stillage, or the like. As the animal proteinaccous material we may employ raw or cooked ground meats, beef or other meat extracts, casein, hydrolyzed casein, egg white, blood, packing house stick liquor, blood albumin, tankage, peptone, or the like. As in the case of the plant proteinaccous material, the animal proteinaccous materials may be at least 3 partially degraded byacid orenzyme action, orboth. .In general it is found that mixtures of the proteinaceous materials and particularly mixtures of the plant and animal proteinaceous materials give superior yields of riboflavin.

The pro rtion of roteinaceous material employed maybesu asto ro defrom0.5 to orh er (as solids) in the medium, although generally from to 5% is found most suitable.

The presence in the medium of a carbon source of the class consisting of the metabolizable carbohydrates and lipids markedly improves the riboflavin yield, particularly when both are employed in admixture. The roportion em toyed may be from 0.1 to about 7%. en carbohy ate alone is employed, it isireferred to add suflictent to give a concentration of 1.0 to 4.0%. Thus metabolizable carbohydrate equivalent to about 1.5 to 2.5% is generally employed in deep tank fermentation, while from about 2.0 to 4.0% carbohydrate is optimal for shaken cultures. Lower levels of carbohydrate cause a corresponding reduction in vitamin synthesis while higher levels, i. e. greater than about 4.0%, although not deleterious, do not correspondingly promote synthesis and consequently are not economical. Various sources of carbohydrate may be used, such as commercial crude and pure glucose, sucrose. maltose, maltose syrups, hydro] and black-strap, high-test, or refiners molasses. With certain of these materials. e. g., some of molasses, it has been found advantageous to mix e carbohydrate as received with an equal weight of water and hydrolyze with dilute mineral acid for to' 30 minutes at a steam pressure'of 15 p. s. i. g. (pounds per square inch gauge) before incorporating it into the medium.

The lipid employed may be an animal fat or oil or a vegetable oil, which for convenience in use, should have a melting point below the temperature at which the propation 0 the organism is carried out. Suitable materials r this pu are corn oil, cottonseed oil, soybean oil, palm oil, olive oil, or the like, and lard, butter, olein, or the like. In general, the proportion of the lipid which is used is preferably in the range from 0.1 to 2.5% when used in the absence of carbohydrate. Similar proportions give improved results when carbohydrate is present, and

ikewise the addition of even small amounts of carbohydrate greatly improves the yields when lipids are the principal carbon source. Thus, in similar media, with 0.1 to 2.5% of lipid present, the addition of 0.1 to 2.5 96 glucose ma increase the yield of riboflavin from 50 to 100%; and wit 2 to 4% glucose present, the addition of 0.1 to 2% lipid likewise greatly increases the riboflavin yield. 0ptimum yields of riboflavin have been secured from media containing 2 to 4% carbohydrate and 0.25 to 1.0% lipid.

We likewise preferably incorporate in the medium an alkali or alkaline earth metal carbonate, as it appears to stimulate the activity of the organism in the direction of an increased riboflavin synthesis. As examples of satisfactory forms of carbonate we mention specifically calcium carbonate, sodium carbonate. potassium carbonate and magnesium carbonate, as well as combinations of these salts. The maximum stimulatory response is produced by adding any of them to give a concentration of from about 0.1 to 1.0%. However, the presence of carbonate in the medium is not essential to secure the eflcct of the addition of propionate in accordance with this invention.

In accordance with our present invention. we further supplement the medium and stimulate the yield of riboflavin with a propionate radical-yielding compound of the class consisting of propionic acid, its calcium, alkali metal and ammonium salts and its lower alkyl esters. Aryl and aryl-alkyl esters, preferably having not more than 9 carbon atoms may also be employed. When such compounds are present in the medium in molar concentrations of from about 0.001 to 0.03, riboflavin production proceeds more rapidly and final yields are substantially increased over those in comparable media lacking the ropionate radical-yielding compound. In media in w ich the organism propagates with negligible or no substantial yields of riboflavin, the incorporation of a propionate .adical-yielding compound has a directive influence, and causes the synthesis of substantial yields of the flavin. Levels of the propionate radical-yielding compound apareciably over 0.03 molar inhibit growth and generally eduoe the riboflavin yield.

For the purpose of illustrating the procedures employed in carrying out the present invention with respect to prepantics: of the medium its handling, inoculation with the organism and p tion of the latter, we set forth a general descri tion 0 an operation employing a medium containing bo plant and animal protemaceous material. Insofar as specific constituents of the medium are referred to in this description, they are merely illustrative, the primary purpose thereof being to set forth generally the conditions employed.

In preparing the medium, a proteinaceous grain processing resi ue or other lant proteinaceous material may be employed. If not 0 a concentration within the desired range, it is preferably diluted with water to give from 0.4 to 3% of solids derived from such residues in the medium. W1thrn this range, the preferred concentrations of the solids from the gram processing residue may vary somewhat. Thus, with a grain alcohol stillage derived from a mixed grain mash, the concentration of the stillage solids may be from 0.75 to 2.25%. With corn steep liquor, a solids concentration in the fermentation medium of from about 0.5 to 3.0% is preferred.

With reference to the animal proteinaceous products, packing house stick liquor or tankage may be used. Partial degradation of the animal protein supplement is desirable for best yields and this can be accomplished, if necessary, by treatment with either acids or enzymes or combinations thereof. The optimum concentration of these products in the final medium ranges from about 0.5 to 4.0 percent depending upon their moisture and content of essential factors. From 1.0 to 2.0 percent stick liquor contaming about 50 percent solids is commonly used.

To prepare a growth medium containing the above ingredients, the proteinaceous grain processing residue or other lant proteinaceous material is first diluted with water, a r which the carbohydrate, animal protein, carbonate and proptonic acid supplements are added. The pH of the mixture is nerally found to be in the range from about 3.? to 4. and is adjusted either prior to or after sterihzatlon by adding soda ash or sodium hydroxide, although other alkalizing mm, such as the and alkaline hydroxides and carbonates of the earth metals, could also be used.

Sterilization is efl'ected by holding the medium preferably at about pH 4.0 to 5.0 under a steam pressure of about 15 to 25 p. s. i. g. for 30 to 60 minutes, or by flash sterilization at 280-300 F. for 3 to 4 minutes, although various other procedures could be used. It has been found, however, that longer periods of sterilization, as well as higher temperature for an equal length of time, are deleterious to riboflavin production and should be avoided.

The initial pH of the medium may vary from about 5.0 to 8.0 although it has been found preferable to adjust the medium before inoculation to a pH between about 5.5 and 7.0. During fermentation the medium at first becomes more acid, i. e., in the neighborhood of pH 4.8 to 5.0, then later rises to a final reaction between about pH 7.5 and 8.5.

In small flasks, aeration of the medium is accomplished by employing volumes of medium equal to about one-fourth the total volume of the flask and placing the flasks in a continuous reciprocating shaking machine. For larger volumes of medium, sterile air is introduced directly and dispersed preferably in a finely divided state within the medium. Perforated pipe or porous stone spargers are suitable for this purpose. Mechanical agitation has been found to improve the efficiency of aeration and tends to reduce the volume of air required. Aeration rates from about 0.05 to 1.0 and preferably 0.05 to 0.5 volume of air pervolume of medium per minute are employed depending upon the dimensions of the fermenter. It should be understood, however, that various other aeration techniques could be utilized to effect aeration of the mash and that our process is not restricted to the particular method mentioned.

The temperature at which fermentation can be conducted may range from about 18' to 35' C. It is preferable, however, to maintain the temperature between 22' and 32' C. for most rapid production and best yields of riboflavin. When the temperature is held below 18' C. growth and vitamin production are retarded, while at temperatures appreciably above 35' C. ribollavin synthesis is reduced.

Inocula are usually prepared by transferring from an actively-growing culture which is carried on an agar slant to a small flask of liquid medium such as peptoneglucose solution. This flask is aerated by shaking and employed after 24 or 48 hours to inoculate a lar volume of the same medium. Aeration is eflected this culture by directly introducin sterile air. After a riod of 24 to 48 hours this c ture is transferred to n stillage medium. We prefer to use a roximately 3% of inoculum by volume, although equ y good results are obtained with inocula from about 0.1 to 5.096- by volume. Larger amounts of moculum have been found to deegress vitamin formation although growth is not ofect Incubation for at least three to four days is commonly required to attain maximum riboflavin formation, and frequently a period of five or six days is found desirable. The length of incubation, of course, is dependent upon such other factors as temperature, pH, availability of carbolgdrate or ligid and other nutrients, etc. and may be as on as 48 ours or as long as one week. When the fermentation is completed, riboflavin recovery can be carried out in any of several ways. The whole fermented medium may be adiiusted to a pH favorin the stability of riboflavin, name y from about 2.5 to 6. evaporated to a syrup, anddried on a drumor spray drier. In this manner all the vitamins and animal growth factors produced by the organism are retained in the final product. if it is desirable to secure higher riboflavin potencies in the final product, the extraneous material may be separated prior to drying by filtration, centrifuging, precipitation, or other known means. If this procedure is followed, complete liberation of riboflavin from the cells of Ashbya gossypii is first brou t about by adjusting the pH to about 2.5' to 6.0 by a ition of acid such ashydrochloric, sulphuric, nitric, or other mmeral acid and heating the culture to a temperature in the neighborhood of 90-150 C. for a period of to 30 minutes. Absorption-elution techniques may also be used to bring about a further purification of riboflavin from the hydrolyzed and clarified liquors.

Our invention may be illustrated more specifically by the following examples:

EXAMPLE 1 A medium was prepared consisting of 50% by volume of stillage obtained from a primary fermentation mash consisting of 86.6% corn, 3.1% rye and 10% barley malt, and also containing 1.0% peptone, 2.0% glucose and 0.1% calcium carbonate. Equal amounts of the medium were placed in fermentation flasks. Sodium propionate was added to duplicate flasks to provide concentrations from 0 to 0.03rnolar. The flasks were adjusted to pH 6.2 and were plugged with cotton and sterilized with steam at a pressure of p. s. i. g. for minutes. After cooling, each flask was seeded with 1.0% of an actively growing culture of Ashbya gossypii. The cultures were incubated for five days at C., during which time the flasks were shaken continuously in a mechanical shaker. The riboflavin yields were determined fluorometrically. In each case, the values given in the table below are the averages of determinations from duplicate flasks. The results were as follows:

Table I Riboflavin Insane Propionlc Acid Concentration, Molar Jml' are sun so as: m 155 4st m 270 M0 30 (Note) In this and the following experiments employing flasks, the flasks were approximately 20% filled with the medium employed.

With larger proportions of propionic acid, the yields of riboflavin are further materially reduced.

EXAMPLE 2 Media were prepared containing varying concentrations of solids from grain alcohol stillage obtained from a primary fermentation mash consisting of 16% corn, 71% milo, 3.2% feed barley and 9.8% barley malt. In each case, the stillage was supplemented with 2% animal stick liquor, 2% glucose an .1% calcium carbonate. 'Ihemedrawereineachcase'adjustedto H6.$bythe addition of small amounts of sodium h xide, where necessary, and divided into equal volumes, which were glaced in equal amount in similar fermentation flasks. or each concentration of stillage solids, propionic acid was added in duplicate flasks to provide a concentration of 0.0! molar and no propionate compound was added to duplicate flasks, which served as controls. The flasks were sterilized at 15 p. s. i. for 20 minutes, and after cooling were inoculated wi 1% of an actively growing culture of A. gossypii. They were then incubated at 28-29 C. for 6 days. Riboflavin was determined fluorometrically on the culture liquors. The values given below are the averages of duplicate flasks.

Table 2 Ribtllavln, Juli.

scu Solids, Percent Without was Pro- Proplonata can ass is an as so sao as so an as as see 440 as ass as us The proportion of stillage solids may be further increased to provide a total content of proteinaceous materral of as high as 10% or even higher, but the riboflavm yield 18 not proportionally increased. However, the cflect of the propionate in stimulating riboflavin synthesis is marked at all proportions of the stillage solids and is apparent in their absence, with only animal protemaceous material present.

EXAMPLE 3 A basal medium was prepared consisting of 50% by volume of gram alcohol stillage obtained from a rimary fermentation mash composed of 88.5% corn, 3) barley and 8.5 barley malt, with 0.1% calcium carbonate and 2.0% of glucose. Equal amounts of the medium were placed in fermentation flasks. Various proteinaceous plant products or products of plant origin were added in amounts of 0.5% or 1% to each of four of these flasks. In each set of four flasks containing identicals media, proptontc acid was added to two of the flasks to provide a final concentration of 0.01 molar, the other two flasks serving as controls. The flasks were adjusted to pH 6.5 by adding sodium hydroxide, adjusted to equal volumes, plugged with cotton and sterilized with steam at 15 p. s. 1. g. for 20 minutes. After cooling, each flask was seeded with 1.0% by volume of an active growth culture of Ashbya gossypii. The flasks were incubated for six days at 28' C., during which time the were staken continuously in a mechanical shaker. e riboflavin yields were determined fluorometrically.

The values given below are the averages of the analyses of duplicate flasks. The results were as follows:

Table 3 muss-m. lI-Iml.

Increase, Medium Supplement, Peromt ith cm H mm Proplonate as: m m m an no or an. am one 400 no sea m an no too son as: so 420 m s04 :04 an are m m are In this and subsequent tables the bol 10 means less than 10 and indicates a substantia ll y negligible yield of riboflavm. The high stimulatory eliect of the proprionate 7 instimula riboflavin eldlsapparentinallinstances intheforeg bi g example.

. EXAMPLE 4 A basal medium was prepared containing 2.0% corn steep liquor, 0.1% calcium carbonate and 0.25% corn oil. Various proteinaceous supplements of plant origin were added at 1.0% concentration to a series of flasks of this medium. In each series four flasks received 2.0% glucose, two of these also receiving 0.01% molar roplonate. Another four received 4.0% glucose an of these, two received 0.01 molar propionate. Propionate was provided as in the previously example. All flasks were adjusted to 6.5 pH, sterilized, and seeded with an actively growing culture of Ashbya sry ii as in the previous example. The flasks were incu at in a mechanical aker as bed in Example 3. Riboflavin yields were fluorometrically on duplicate flasks and averaged. The results were as follows:

In the series of tests presented in this example in which the proteinaceous material in the medium was entirely of plant origin, the highly stimulatory eflect of propionates on riboflavin yield was again demonstrated at varying carbohydrate levels, even in media wherein, without propionate, insignificant yields were secured.

EXAMPLE 5 Media were prepared, each containing 1.5% stilla e solids of the same character as were employed in e media of Example 2, 2% animal stick liquor, 0.1 calcium carbonate and varying proportions of different carbohydrates as indicated below. Equal amounts of the media were placed in similar fermentation flasks. acid was added to duplicate flasks to provide a. concentration of 0.01 molar and duplicate flasks with no addition of the propionste compound were likewise run. The flasks were sterilized, inoculated with A. or pit and incubated as described in Exam le 1. RI avin yields were determined fluorometrical y on duplicate flasks and averaged. The results were as follows:

EXAMPLE 6 Media were prepared containin varying proportions of solids from corn steep liquor an of animal stick liquor. In each case the medium contained 2% glucose and 0.1%

Propionic [all calcium carbonate. Thetestswereconducted aslnBxample 2 above. The results were as follows:

Table 6 Riboflavin ale-M eroen t Llq With Jan.

I" Percent 0.01 H

Propi Pm onate m 1.0 we as: 27 2. 0 see us 216 1.0 5 m 18H 1. 5 556 no IN 20 sec use 320 1.0 510 m m l. U 838 2 I 8.0 as!) too no EXAMPLE 7 Media were prepared with difl'erent proteinaceous grain residue solids and with dilferent animal protein materials, asindicated in the ensuing table. In each case the medium contained 2% glucose and 0.1% calcium carbonate. The tests were conducted as in Example 2 above. The results were as follows:

Media were prepared in which the proteinaceous material present was derived solely from animal sources. The basal medium was an aqueous solution containing 0.1% calcium carbonate, 0.25% dibasic potassium. phosphate, 2% glucose and 0.25% corn oil. Various animal proteinaceous substances, as indicated in the followin table, were added in proportions ranging from 0.25 to 1% to each of the series of four flasks containing the foregoing basal medium. 0.01 molar propionate was added to two of the four flasks in each group. The flasks were then adjusted to a pH of 6.5, and sterilized in the same manner as in Example 3 above. After cooling, each flask was inoculated with 1.0% by volume of an actively growing culture of Ashbya gossy ii. The flasks were incubated for six days at 28' C., during which time they were shaken continuously on a mechanical shaker. The riboflavin was determined fluorometricall as in the grevious examples. The values given in the to lowing ta le are the average of the analyses of duplicate flasks. The results are as follows:

Table 8 Riboflavin, gJml.

Increase Mum Without With .01. M gJ'ml.

Pro- Proplonate plonate 0. animal peptcne 328 M2 1.0 anlrurilsllafeptone 250 135 0.5 anl peptone+.25 animal atlckl uor as an as 1.0 animal peptone+.25 animal 0 :tlckl uonuaanwinsiu--. 03 174 81 animal as ex- .E 02 214 172 0.6 animal peptona+0.5 casein 308 183 It is thus apparent, as illustrated in Examples 5 through 8. as well as in Examples 1 and 2, that the highly stimulatory effect of propionates on riboflavin production are secured where the proteinaceous constituents of the media are derived in part or in whole from animal sourcesas well as when derived entirely from plant sources. Thus proteinaceous materials of any character may be emplayed. as the benzyl, phenylethyi, pheaylethyl and tolyl proplonates.

m EXAMPLE 11 ployed in media for use in carrying out the present inven- Tests showed that in a medium with only propionic acid i it lpzopionates in the absence of a carbon source, s yagossypi togrow. Furthertestsweremade EXAMPLE 9 to compare the effects of added propionate radical-yield- In another example, as in the previous example, both ing compound on the bi thesis of riboflavin by Ashbya a metabolizable carbohydrate and a lipid were present It! gossypii with those resulting from a carbon source, such in the medium and various combinations of animal and as metabolizable carbohydrate. In these tests the media protcinaceous materials were employed. 'lhe basal meemployed contained 1.6% grain stilla solids of the dium was an aqueous solution containing 2.0% glucose, character used in Example 2, with 2. animal stick 0.5% corn oil. 0.5% dipotassium phosphate and 0.1% liquor and 01% calcium carbonate. Three different levels calcium carbonate. Animal and plant proteinaceous maof glucose were employed, L595, 2.0% and 2.5%. At terial were added as indicated in the following table. each level comparative tests were made with equal addi- With each combination of supplements, four flasks were tions of glucose and of propionic acid. The tests were Ere aretfi andh0.0l% 15:01:; ptrsopionatedwas added it; $50 carried out as in Example 2. The results were as follows:

as s o eac set. e as were a juste to sterilized, inoculated with an actively growing cu ture of 20. Tab! 11 Ashbya gossypii and incubated in the same manner as in the preceding example and in Exam le 3. The riboflavin Glucose Level Glucose a? Riboflavin was determined fluorometrically. T e values given in the cm Added A vigi following table represent the average analyses of dupli- PM Pereen w eate flasks in each instance.

La 9 cum g mbmvm Film] 2'0: 44o r .0 W "star as .s suppkm" Without With .01 3

D v f? gig: a'sI "BIG-ii 1,150 no 2 corn steep liquor-+1 animal stlck 3 These results clearly show the extraordinary eflects of it nor 5g 33 the supply of the proplonate radical in the medium upon imbfiarstaruaanaiarmaa:tr 11 no 39 s Produwon by orlflmsmiiin iph? 121% idih'isaz: 33 3 EXAMPLE 12 lllilf; iti+i ammiii$iiftaii: 3 late a 1300 gallon fermenter equipped with an aerating 1 when gluten-+1 m new, m 375 136 device and mechanical agitator were pumped 450 gallons of stillage, ttgbstaltgtg from gopgganary fermentation mash ting o corn, rye and 11.7% barley From the foregoing examples it is apparent that the corms stimulatory effect of propionates on riboflavin synthesis & gallons 3 one and fifty by Ashbya gossypii is secured when the carbon source in gggg g g g zg wa 2 2223 22;"? g i; the medium is entirely carbohydrate or a mixture of carminutes with Steam 8 a i of 15 s H bohydrate and lipid. It is likewise exerted when the d p s1 e p carbon source is a lipid in the absence of carbohydrate. urmg sterilization was 4.1. After sterilization, l4 pounds Thus com on cottonseed on when oil palm oil olive of sodium carbonate and 5.25 unds propionic acid WhlCh oil, lard, butter, olein and oiher natural iats and oils or 0" is f stcnhzgld gf g water solution were added mixtures thereof, in proportions as hereinbefore set forth, b [ought e p to The total volume-of when employ in media with plant or animal pmteinace tum ecause of steam condensation and the addition of mm material Show an augmented riboflavin yield in the neutralizing agent was approximately 1,000 gallons. The presence Of impionic acid or propionates medium was cooled to 29 C. and seeded with 40 gallons of a 24-hour culture of Ashbya gossypii grown on 1.0 EXAMPLE l0 perceit pgptons1 and l.0fpe)rlcut gllucose. S terile air was Tests were made to compare the effectiveness of a 9 at 6 {ate 0 yo of Per i lower alkyl propionate, such as butyl propionate with g. gg i g l a' alts z was that of propiomc acid in increasing the yield of riboa f t? g?" and flavin with Ashbya gassypii. Media were prepared conmg e course 0 a an own belowtaining 1.5% stillage solids of the character described in Table I2 connection with Example 2, 1.0% peptone, 2% ucose and 0.1% calcium carbonate. The tests were earned out mboflmn as-described in connection with Example 2 except that, 11mm ommmmmn pH m when using butyl propionate, due to its volatility, it was 05 "Juli. sterilized separately by Seitz filtration and added to the medium after it was sterilized and cooled. The results 10... .5 m were as follows: g g-g &

as "I Table 10 g 2% a ddll Medl tii'i tir 933' I h D A Add it ive gJml'. ll/m1 EXAMPLE 13 We have found that the enhancement of riboflavin pro- 0 m M a ductton by the am Ashbya gossypii is stimulated to 1 m an extraordinary egree by incorporating into the medium, 0.01 M 571 as! In addition to a proplonate radical-yielding material, a use M 495 15 small proportion, suitably 0.25 to 1.5% of yeast substancecontaining material, such as, for example, dried brewer's Other lower alkyl propionates may be employed, such l ye st extract. Bllcbllel' yeast i Y fflmemaas the ethyl, propyl or amyl esters, to supgly the protlou residues or the like. The addition of such yeast pionate radical to elfect the stimulation of ri flavin prosubstance-containing materials to some media suitable for duction of the organism. Aryl and arylalkyl propionates the propagation of Ashbya gossypii, in the abcenee of the having not more than 9 carbon atoms may also be em- 86 proptonate radical, may not result in any increase in yield of riboflavin and may even result in a diminution of yield, although in other media which do not contain fermentation residues such as the synthetic medium described in the application of Van Lanen et al., Serial No. 77,094, filed Fe ary 21, 1944, such additions augment the yield of ribo avin. In all cases, when both a yeast substancecontaining material and a propionate radicalyielding material are incorporated in the medium, the yield of riboflavin is very greatly increased beyond that secured by the propionate alone. This is illustrated in this and the followin example.

A asal medium was prepared consisting of 50% by volume of stillagc obtained from a primary fermentation of a mash of the same composition as that referred to in Example 3 above, 0.1% calcium carbonate, 1.0% animal stick liquor and 2.0% glucose. Dried brewer's yeast was added in difierent proportions to each of four flasks of this medium and in two of each series of four, .01 molar propionate was supplied. The media in all flasks was adjusted to a pH of 6.5, sterilized, inoculated with Ashbya goss'ypil' and incubated in the same manner as in Examle 3. The riboflavin was then determined fluorometricalfy. The values given in the following table in each case represent the average of the analyses of duplicate flasks.

This example was carried out in the same way as the p example, except that the medium used was composed of 2% corn steep liquor, 0.1% calcium carbonate, 1.0% animal peptone, 0.25% corn oil and 2.0% glucose. Dried brewer's yeast in proportzons of 0.5% and 1.0% were added to each of a series of four flasks and in each of these series, two of the flasks received 0.01 molar propionate. The procedure as to ad ustment of pH, sterilization, inoculation and incubation was as in the preceding example. The resulting riboflavin yield, as

given in the table below, in each case represents the average analyses of duplicate flasks.

In the foregoing Examples 8, 9, l3 and 14 the propionate was supplied as propionic acid.

Table 14 v Riboflavin Yield,

wi In Hedlum Supplement m with! with 0.011!

Preptenate Pmptonats Nona 454 m In 0.5 W I Hit- M II. m mama $.34 in: m 1.1a can In the foregoing Examples 13 and 14, for convenience, the yeast substance-containing ma was added in the form of dried brewer's yeast. ,When added in the form of yeast extract, yeast uiee, yeast fermentation residues in similar proportions, on dried solids, similar results are secured.

it will be noted in fore ing Examples l3 and 14 that in each case the yield riboflavin was very markedly increased by the conjoint action of the yeast substance and the propionate. In Example 13, for male in which the ma medium included constituents rived from yeast fermentation, a has increase in yield of 522 gJml. was secured by the dition of the prozsionate alone. with added yeast in proportionstfrom 0. to l.0% in the medium, although the 1d of riboflavin was actuall reduced in the absence 0 added propionate,

the yield with 0.01 M pro ionate was even more greatly increased from 172 to 6 agJml. In Example 14, in wbichnomaterialsderivedfromyeastfeamentationwere present in the basal medium, some increase in riboflavin yield was secured by the addition of the yeast in the absence of propionate. Thus with 0.5% yeast the increase was 56 ag ./ml. andwith 1% yeast, 52 agJml. With 0.01 M propionate, without added yeast, an increase in yield of .g./ml. was secured. With both propionate and yeast however, an enormously greater increase over the yield from the basal medium alone was secured,

being 392 gJml. with 0.5% yeast and 712 agJml. with 1.0% yeast.

Although in many of the preceding examplesthe propionate radical-yielding compound employed, for reasons of convenience, was proplonic acid or a pro ionate, it has been found that like effects are produ in all media with other propionate radical-yielding compounds of the class above described, including the alkali and alkaline earth metal propionates, ammonium propionate and the lower allg'l, aryl and arylalkyl esters of propiomc acid. The e ect of ropionate in increasing the riboflavin yield is secured with additions of 0.001 to 0.03 molar concentrations of the propionate radicalyielding compounds in all media capable of sustaining the growth of Ashbya gasrypii', even in media in which, in the absence of a propionate supglement, no substantial quantities of riboflavin are pro need. The addition of yeast substance-containing material in proportions of 0.1% to 5.0% in all cases greatly increases the riboflavin yield in the presence of these proportions of propionate radical-yielding compounds.

in its propagation in accordance with the present invention the organism Ashbya gossypii synthesizes, besides riboflavin, other vitamins and growth producing factors of value, as more fully set forth below. The riboflavin may be separated out from the final culture liquor, as may other vitamins and materials of value, if desired. However, the entire product of fermentation may be utilized beneficially, for example, as a growth promoting supplement in animal feeds. This is illustrated by the following example.

EXAMPLE 15 Final culture liquor prepared similar to that described under Example 12 was acidified to pH 5.0, evaporated to a syrup, and dried on a drum drier. The dry product was examined for its content of chick growth factors by the following procedure. A starter ration was compounded which contained adequate amounts of all the nown dietary factors as recommended by the National Research Council Committee on Animal Nutrition. This ration was divided into three lots, the first of which was not sueplplemented and served as a control. The second receiv 5 percent distillers' dried solubles and the third received 4.95 percent of distillers' dried solubles and 0.05 percent of dried Ashbya goss'ypii culture. From 60 to 65 one-day-old chicks were placed on each ration and the three groups were maintained under'comparable conditions during a six-week test period. At the end of this time the chicks were weighed and the weights averaged. The average weights after equalizing the numbers as to In Exam le 15 it is demonstrated that our final fermentation pr act, when utilized in animal rations, provides both riboflavin and unidentifled growth stimulating factors. The unidentifled factors simulate in their growth promoting properties the effects commonly obtained by adding fish or meat products to the ration. However, use 0 the product is not limited to animal feeds since it is obvious that all or a part of the riboflavin might be separated and employed in harmaceutical preparations, food products, etc. and e residue incorporated into feed products.

It has been found that, in carrying out the present invention, the so-called Lactobacillus Bulgaricus Factor, also called LBP, is synthesized in unusually large amounts, and appears in the residue of the fermentation process employed as a food supplement, as above set forth. This creases factor was first reported by Williams, Hofi-Iorgenscn and Snell, J. Biol. Chem, vol. 177, pages 933-940 (1949).

concentrates have been found to increase the weight gain of chicks and rats and this factor may be an important constituent of the ration of various animals. Ashbya gossypii, fermented under the conditions of the present invention, lgroduces from twelve to over twenty times as much of e LBF as do related yeast and yeastlike organisms, such as Candida crusoides (arborea), Mycotorula lipolytica, Saccharaomyces cercvisia and Torula utilis.

While fermentation with Arhbyagossypii ishighly effective in the biosynthesis of riboflavin, it also substan tially increases the yield of other vitamins, such as pantothenic acid, niacin, pyridoxin, folic acid, biotin and paminobenaoic acid. As these substances are likewise carried into the residue product, they likewise enhance greatly its value as an animal feed supplement.

It is apparent that in all media suitable for the efiective ropagation of Ashbya gossypii with biosynthesisof ribo vtn, the addition of a propionate radical-yielding compound has a marked, unexpected stimulatory eflect upon the riboflavin Id, and that, when further supplemented by yeast su ance-yieldmg materials, a marked increase in this stimulatory eliect is secured. Although the invention has been illustrated in connection with various specific examples thereof, it is to be understood that the details of these examples shall not be regarded as limitations upon the scope of the invention, except insofar as included in the accompanying claims. 7

We claim: a

l. The process wherein the ascomycete Ashbya gossypil is propagated under aerobic conditions in a liquid medium containing as principal ingredients proteinaceous material and a carbon source from the class consisting of metabolizable carbohydrates and lipids, the step of stimulating the synthesis of riboflavin by the organism by mcorporating in said medium a propionate radical-yteldmg compound of the group consisting of propionic acid, its. salts and lower alkyl, aryl and arylalkyl esters, said propionate radical-yielding compound being present in an amount corresponding to a molar concentration of 0.001 to 0.03.

2. 'lhe process of claim l wherein the carbon source in the medium is a mctabolizable carbohydrate.

3. The process of claim 1 wherein the carbon source in the medium is a lipid.

4. The process of claim 1 wherein the carbon source is both a metabolizable carbohydrate and a li id.

5. The process wherein the ascornycete Ash ya gassypu is propagated under aerobic conditions in a liquid medium contaming as principal ingredients proteinaceous material and a carbon source from the class consistin of metabolizable carbohydrates and lipids, the step 0 stimulating the synthesis of riboflavin by the organism by mcorporating in said medium a yeast substance-containing material and a propionate radicalyielding compound of the group consisting of propionic acid, its salts and lower alkyl esters, said propionate radical-yielding compound being present in an amount corresponding to a molar concentration of 0.001 to 0.03.

6. The process of claim 5 wherein the carbon source in the medium is a metabolizable carbohydrate.

7. The recess of claim 5 wherein the carbon source in the m ium is a lipid.

8. The process of claim 5 wherein the carbon source is both a metabolizable carbohydrate and a livid:

9. The process for producing riboflavin compris ng the steps of propagating the ascomycete Ashbya gpsryph under aerobic conditions in a liquid medium containing as prmcipal ingredients a proteinaceous material, a carbon source from the class consisting of mctabolizable carboh drates and lipids, a carbonate of the group consisting of e carbonates of the alkali and alkaline earth metals, and a propionate radical-yielding compound of the group consistlng of propionic acid, its salts and lower alkyl esters, said propionate radical-yielding compound being present in an amount corresponding to a molar concentration of 0.001 to 0.03.

10. The process of claim 9 wherein the carbon source is a metabolizable carbohydrate.

11. The process of claim 9 wherein the carbon source is a lipid.

12. The process for producing riboflavin comprising the steps of propagating the ascomycete Ashbya gorsypti under aerobic conditions in a liquid medium containing as principal ingredients a proteinaceous material, a carbon source from the class consisting of metabolizable carbohydrates and lipids, a carbonate of the group consisting of the carbonates of the alkali and alkaline earth metals, a yeast substance-containing material, and a propionate radical-yielding compound of the group consisting of propromo acid, its salts and lower alkyl esters, said propionate radical-yleldmg compound being present in an amount corresponding to a molar concentration of 0.001 to 0.03.

13. '1 he process for producing riboflavin comprising the steps of propagating the ascomycete Ashbya gossypii under aerobic conditions in a liquid medium containing as principal ingredients plant proteinaceous material, a carbon source from the class consisting of metabolizable carbohydrates and lipids, a carbonate of the group consisting of the carbonates of the alkali and alkaline earth metals, and a propionate radrcabyrelding compound of the group consistingof propionic acid, its salts and lower alkyl esters, satdpropionate radical-yielding compound being present in an amount corresponding to a molar concentration of 0.001 to 0.03.

14. The process for producing riboflavin comprising the steps of propagating the ascomycete Ashbyc gossypii under aerobic conditions in a liquid medium containing as principal ingredients an animal proteinaceous material, a carbon source from the class consisting of metaboiizabie carbohydrates and lipids, a carbonate of the group consisting of the carbonates of the alkali and alkaline earth metals, and a propionate radical-yielding compound of the group consisting of propionic acid, its salts and lower alkyl esters,

said propionate radical-yielding compound being present" in an amount corresponding to a molar concentration of 0.001 to 0.03. 7

15. The process for producing riboflavin comprising the steps of propagating the ascomycete Ashbya gossypii under aerobic conditions in a liquid medium containing as principal ingredients a plant proteinaceous material, an animal protcinaceous material, a metabolizsble carbohydrate, a carbonate of the group consisting of the carbonates of the alkali and alkaline earth metals, and a propionate radical-yielding compound of the group consisting of propionic acid, its salts and lower alkyl esters, said propionate radical-yielding compound being prcsentin an amount corresponding to a molar concentration of 0.001

16. The process for producing riboflavin comprising the steps of propagating the ascomycete Ash bya gossypii under aerobic conditions in a liquid medium containing as principal ingredients a plant proteinaccous material, an animal proteinaceous material, a metabolizable carbohydrate, is carbonate of the group consisting of the carbonates of the alkali and alkaline earth metals, a yeast substance-containing material and a propionate radical-yielding compound of the group consisting of propionic acid, its salts and lower alkyl esters, said propionate radical-yielding compound being present in an amount corresponding to a molar concentration of 0.001 to 0.03.

17. The process for producing riboflavin which comprises the steps of propagating the ascomycete Ashbya gosiypii under aerobic conditions in a liquid medium containing as principal ingredients as proteinaceous grain processin residue, an animal proteinaceous material, a metabolizable carbohydrate, a carbonate of the group consisting of the carbonates of the alkali and alkaline earth metals, and a propionate radical-yielding compound of the group consisting of propionic acid,.its salts and lower alkyl esters, said propionate radical-yielding compound being present in an amount corresponding to a molar concentration of 0.001 to 0.03.

18. The process for producing riboflavin which comprises the steps of propagating the ascomycete Ashbya gossypit under aerobic conditions in a liquid medium containing as principal ingredients a proteinaceous grain processing residue, an animal protemaceous material, a metabolizable carbohydrate, a carbonate of the group consisting of the carbonates of the alkali and alkaline earth metals, a yeast substance containing material and a propionate radical-yielding compound of the group consisting of propionic acid, its salts and lower alkyl esters, said propionate radical-yielding compound being present in an amount corresponding to a molar concentration of 0.001 to 0.03.

19. The process for producing riboflavin comprising the steps of propagating the ascomycete Ashbya gosrypii 86 under aerobic conditions in a liquid medium containing as principal ingredients grain alcohol stillage solids, an animal proteinaceous material, a metabolizable carbohydrate, a carbonate of the group consisting of a carbonate of the alkali and alkaline earth metals, and a propionate radical-yielding compound of the group consisting of propionic acid, its salts and lower alkyl esters, sald propionate radical-yielding compound being present lll an mount corresponding to a molar concentration of 0.001 to 0.03.

20. The pi )cess of claim 19 wherein the animal proteinaceous material is animal stick liquor.

21. The process of claim 19 wherein the animal proteinaceous material is peptone.

22. The process of claim 19 wherein the metaboltzable carbohydrate is glucose.

23. The process of claim 19 wherein the metabolizable carbohydrate is maltose.

24. The process of claim 19 wherein the metabollzable carbohydrate is sucrose.

25. The process for producing riboflavin comprising the steps of propagating the ascomycete .dshbya gossypii under aerobic conditions in a liquid medium containing as principal ingredients grain alcohol stillage solids, an animal proteinaceous material, a metabolizable carbohydrate, a carbonate of the group consisting of a carbonate of the alkali and alkaline earth metals, a yeast substancecontaining material and a propionate radical-yielding compound of the group consisting of propionic acid, its salts and lower alkyl esters, said propionate radical-yielding compound being present in an amount corresponding to a molar concentration of 0.001 to 0.03.

26. The process for producing riboflavin comprising the steps of propagating the ascomycete Ashbya gossypri under aerobic conditions in the liquid medium containing as principal ingredients corn steep liquor, an animal proteinaceous material, metabolizable carbohydrate, a carbonate of the group consisting of the carbonates of the alkali and alkaline earth metals, and a propionate radicalyielding compound of the group consisting of propionic acid, its salts and lower alkyl esters, said propionate radical-yielding compound betng present in an amount corresponding to a molar concentration of 0.001 to 0.03.

27. The process of claim 26 wherein the animal proteinaceous material is animal stick liquor.

28. The process of claim 26 wherein the animal proteinaceous material is peptone.

29. The process of claim 26 wherein the metabolizable carbohydrate is glucose.

The process of claim 26 wherein the metabolizable carbohydrate is maltose.

31. The process of claim 26 wherein the metabolizable carbohydrate is sucrose.

32. The process for producing riboflavin which comprises the steps of ropagating the ascomycete Ashbya gossypii under aero ic OOlldlllOllS in a liquid medium containing as principal ingredients from 0.4 to 3% of plant proteinaceous material, 0.5 to 4.0% of animal proteinaceous material, 1.5 to 4.0% of mctabolizable carbohydrate, 0.1 to 1.0% of a carbonate of the group consisting of the carbonates of the alkali and alkaline earth metals, and 0.001 to 0.03 molar concentration of a propionate radical-yielding compound of the group consisting of propionic acid, its salts and lower alkyl esters.

33. The process of producing riboflavin which comprises the steps of propagating the'ascomycete Ashbya gossypii under aerobic conditions in a liquid medium containing as principal ingredients 0.75 to 2.25% grain alcohol stillage solids, 1.0 to 2.0% animal stick liquor, 1.5 to 4% metabolizable carbohydrate, 0.1 to 1.0% calcium carbonate and 0.001 to 0.03 molar concentration of a propionate radical-yielding compound of the group consisting of propionic acid, its salts and lower alkyl esters.

34. The process for producing riboflavin comprising the steps of propagating the ascomycete Ashbya gossypii under aerobic conditions and liquid medium containing as principal ingredients 0.5 to 3.0% corn steep liquor solids, 1.0% to 2.0% animal stick liquor, 1.5 to 4.0% metabolizable carbohydrate, 0.1 to 1.0% calcium carbonate and 0.001 to 0.03 molar concentration of a proionate radical-yielding compound of the group consistmg of propionic acid, its salts and lower alkyl esters.

35. The process of producing riboflavin which comprises the steps of ropagatin the ascomycete Ashbya gassypil under aero ic conditions in a liquid medium containing as principal ingredients 0.5 to proteinfat aceous material, from 0.1 to 7% of a carbon source from the groug consistin of metabolizable carbohydrates and liquids, .1 to 1.0 o of a carbonate from the group consisting of the alkali and alkaline earth metal carbonates, and 0.001 to 0.03 molar concentration of a propionate radical-yielding compound of the roup consisting of propionic acid, its salts and lower a kyl esters.

36. The process of producing riboflavin which comprises the steps of propagating the ascomycete Ashbya gqssypii under aerobic condition in a liquid medium contaming as principal ingredients 0.5 to 10% proteinaceous material, from 1 to 4% of metabolizable carbohydrate and from 0.1 to 2.5% lipid, 0.1 to 1.0% of a carbonate from the group consisting of the alkali and alkaline earth metal carbonates, and 0.001 to 0.03 molar concentration of a propionate radical-yielding compound of the group consisting of propionic acid, its salts and lower alkyl esters.

37. The process for producing riboflavin comprising the steps of propagating the ascomycete Ashbya gossypii under aerobic conditions in a liquid medium containing as principal ingredients from about 0.75 to 2.25% of plant proteinaceous material, from about 0.5 to 4.0% of animal proteinaceous material, a metabolizable carbohydrate, a carbonate of the group consisting of the carbonates of the alkali and alkaline earth metals, and a propionate radical-yielding compound of the group consisting of propionic acid, its salts and lower alkyl esters.

38. The process for producing riboflavin comprising the steps of propagating the ascomycete Ashbyn gossypii under aerobtc conditions in a liquid medium having an initial pH between about 5.0 and 8.0 and containing as principal ingredients proteinaceous materal, carbon source material from the group consisting of metabolizable carbohydrates and lipids, a carbonate of the group consisting of the carbonates of the alkali and alkaline earth metals, and a propionate radical-yielding compound of the group consisting of propionic acid, its salts and lower alkyl esters, sai propionate radical-yielding compound being present in an amount corresponding to a molar concentration of 0.001 to 0.03.

39. The process of claim 38 wherein a small proportion of yeast substance-containing material is incorporated in the medium.

40. The process for producing riboflavin comprising the steps of propagating the ascomycete Ashbya gossypii in a liquid medium containing as principal ingredients a plant proteinaceous material, an animal proteinaceous material, a metabolizable carbohydrate, a carbonate of the group consisting of the carbonates of the alkali and alkaline earth metals, and a propionate radical-yielding compound of the group consisting of propionic acid, its salts, and lower alkyl esters, said proptonate radicalyielding compound being present in an amount corresponding to a molar concentration of 0.001 to 0.03, and aerating the medium at a rate from about 0.05 to 1.0 volume of air per volume of medium r minute.

41. The process for producing ribo avin, which comprises the steps of propagating the ascomycete Ashbya gossypii in a liquid medium containing as principal ingredients a plant proteinaceous material, an animal proteinaceous material, a metabolizable carbohydrate, a carbonate of the group consisting of the carbonates of the alkali and alkaline earth metals, and a propionate radicalyielding compound of the group consisting of propionic acid, its salts and lower alkyl esters, said propionate radical-yielding compound being present in an amount corresponding to a molar concentration of 0.001 to 0.03, at a temperature from about 18' to about 35' C., and aerating the medium at a rate from about 0.05 to 1.0 volume of air per volume of medium per minute.

42. The process for producing riboflavin which comprises the steps of propagating the ascomycete Ashbya gossypii in a liquid medium adjusted initially to a pH between about 5.5 to 7 and containing as principal ingredients a plant proteinaceous material, an animal proteinaceous material, a metabolizable carbohydrate, a carbonate of the group consisting of the carbonates of the alkali and alkaline earth metals, and 0.001 to0.03 molar concentration of a propionate radical-yielding compound of the group consisting of propionic acid, its salts and lower alkyl esters, at a temperature between about 22' and about 32' C., and aerating the medium at a rate from about 0.1 to 0.5 volume of air per volume of medium 35 per minute.

43. The process of claim 42 wherein a ar nall proportion of yeast substance-containing material is incorporated in the medium.

44. In the production 01 riboflavin by aerobic fermentation of a nutrient medium with the ascomycete Ashbya gassypii, the improvement which comprises conducting the fermentation in the presence of a substance yielding a propionate radical in the range of molar concentration of 0.001 to 0.03.

45. The process as defined in claim 44 wherein the range of molar concentration of the propionate radicalyielding substance is from 0.005 to 0.02.

References Cited in the tile of this patent UNITED STATES PATENIS 2,098,200 Stiles -..s; Nov. 2, 1937 18 2,202,161 Miner May 28, 1940 2,297,671 Yamasaki Sept. 29, 1942 2,374,503 Rudert Apr. 24, 1945 2,400,710 Piersma May 21, 1946 2,445,128 Tanner July 13, 1948 OTHER REFERENCES MWaltgr and Coppock, 1928, A. Chem. Soc. 1., pages 8 8 Sehopfer, Helvetica Chimica, Acta V, XXVII, pages 1017-1032.

Porter, Bacterial Chemistry, 1946, Wiley, page 812.

Wickerham et al., Arch. Biochem., vol. 9, No. 1, January 1946, pages 95-98.

Williams et al., Iour. Biol. Chem., vol. 177 (1949), pages 933-940. 

1. THE PROCESS WHEREIN THE ASCOMYCETE ASHBYA GOSSYPII IS PROPAGATED UNDER AEROBIC CONDITIONS IN A LIQUID MEDIUM CONTAING AS PRINCIPAL INGREDIENTS PROTEINACEOUS MATERIAL AND A CARBON SOURCE FROM THE CLASS CONSISTING OF METABOLIZABLE CARBOHYDRATES AND LIPIDS, THE STEP OF STIMULATING THE SYNTHESIS OF RIBOFLAVIN BY THE ORGANISM BY INCORPORATING IN SAID MEDIUM A PROPIONATE RADICAL-YIELDING COMPOUND OF THE GROUP CONSISTING OF PROPIONIC ACID, ITS SALTS AND LOWER ALKYL, ARYL AND ARYLALKYL ESTERS, SAID PROPIONATE RADICAL-YIELDING COMPOUND BEING PRESENT IN AN AMOUNT CORRESPONDING TO A MOLAR CONCENTRATION OF 0.001 TO 0.03. 